Int. J. Morphol., 33(1):100-104, 2015.

Histological and Morphometric Analysis of Testes of Adult ( coturnix japonica) of

Análisis Histológico y Morfométrico de Testiculos de Codorniz Adulta (Coturnix coturnix japonica) de Bangladesh

Subrata Kumar Shil*; Md. Abul Quasem* & Mohammad Lutfur Rahman*

SHIL, S. K.; QUASEM, A. & RAHMAN, M. L. Histological and morphometric analyses of testes of adult quail (Coturnix coturnix japonica) of Bangladesh. Int. J. Morphol., 33(1):100-104, 2015.

SUMMARY: A histological and morphometric study was conducted to evaluate the seasonal testicular variations in the adult Japanese quail (Coturnix coturnix japonica) in Bangladesh. The weight of and testes, volume, length, circumference, seminiferous tubule diameter, circumference and height of germinal epithelium of testes were measured. Sections of testes obtained from studied birds were stained with H&E and images were taken by J software. Weight and volume of paired testes of significantly heavier (P<0.05) in long day period of summer and rainy season (March – September) than the short day period of autumn and winter (October – February). Seasons had no significant effect on live weight gain of quails. The weight and diameter of testes or height of the germinal epithelium of the seminiferous tubule were positively correlated (r=0.84; r=0.88). Spermatogenic activity was pronounced during summer and rainy season than that of autumn and winter (P<0.05).

KEY WORDS: Japanese quail; Morphometry; Testis; Season; Day length; Temperature.

INTRODUCTION

Reproduction of birds depends on photoperiod, in temperate zone species (Wingfield et al., 1992; Ball, rainfall, humidity, temperature and food as well as intrinsic 1993; Cockrem, 1995; Hahn et al., 1997). Bangladesh has factors (Lake, 1981; Lofts & Murton, 1968). In temperate a subtropical monsoon climate characterized by wide zones, seasonal day length variation serves as the primary seasonal variations in rainfall, moderately warm role of timing reproductive periodicity (Lofts & Murton). temperatures, and high humidity and there is a little The majority of species outside the tropics rely on a food research work to assess the effect of different factors on resource for their young that become available for a morphometry of testis. Therefore, this study was conducted predictable period each year. The length of the breeding to evaluate the effect of season and daylights on season varies between species, but in the vast majority, morphometry of testis of quail in Bangladesh. the breeding season is asymmetrical with changes in photoperiod. When mature male coturnix were exposed to short photoperiods (6L: 18D) a rapid testicular weight MATERIAL AND METHOD reduction was recorded (Follett & Farner, 1966). Reduction of testicular weight in the common coturnix, exposed to short days (8 h of light), was more rapid in a colder than in Collection of birds. The study was performed on 120 a warmer environment (Wilson et al., 1971). The two most sexually matured domestic quails (Coturnix coturnix critical features in the seasonality of an environment are japonica) weighing average 119.15±7.46 g (range 110 g the amplitude of the seasonal fluctuations and the precision to 130 g). The quail was routinely collected from a with which these fluctuations occur each year (Wingfield recognized farm of Chittagong Metropolitan area, et al., 1992). The physiological processes that underlie the Bangladesh during different seasons of the year. Forty timing of breeding in birds have been studied extensively quails were collected during summer (March-June), 30

* Department of Anatomy and Histology, Faculty of Veterinary Medicine, Chittagong Veterinary & Sciences University, Zakir Hossain Road, Khulshi, Bangladesh. 100 SHIL, S. K.; QUASEM, A. & RAHMAN, M. L. Histological and morphometric analyses of testes of adult quail (Coturnix coturnix japonica) of Bangladesh. Int. J. Morphol., 33(1):100-104, 2015.

during rainy (July- September), 20 during autumn RESULTS (October-November) and 30 during winter (December- February). The birds were euthanized (30 mg/kg bw/im) and the testes were collected from each individual . Morphometry of testes of quail by season and length of day light. Mean live weight of the Japanese quail (n=120) Gross morphometry. After separation of testis, specimen was 119.15±7.46 g. Average paired testicular weight, volume, was subjected to gross anatomical measurement. Volume length and circumference were significantly higher in summer of testes was measured by Archimedes’ principle of water and rainy seasons than in autumn and winter (P=0.02). The displacement using normal saline (0.85% NaCl) and average weight of testes was significantly heavier (3.17±0.66 results were recorded in cubic milliliters (Bath & g) during long day period (summer and rainy season) than Chaudhari, 2002). Length of testes was measured by that of short day period (1.27±0.28 g) (P=0.03) (Fig. 1). vernier digital calipers (0-150 digital caliper, Shinko Regardless of seasons positive correlation was recorded Denshi Co. Ltd, Japan). Relative testis weight was between the day length and paired testicular weight (r=0.82). measured according to formula as follows: (weight of However, paired testes weight were about 2.5 fold higher in paired testes / body weight) ¥ 100 (Orlu & Egbunike, summer than in winter. Relative testes weight was higher in 2010). summer (2.75±0.50%) and rainy season (2.45±0.44%) than the autumn (1.05±0.22%) and winter (1.13±0.29%). Tissue processing. Sampled testes were immediately immersed in Bouin’s fixative and submitted to prefixation for approximately 20 minutes. Longitudinal sections of testes were then made and sections were immersed in histological fixatives for 24 h in order to complete fixation. Small testis fragments were used for the routine histology such as dehydration, clarification and paraplast embedding and stained with hematoxylin-eosin (HE).

Histomorphometry. A total of 12 testes from quails were collected during summer, 9 during rainy, 6 during autumn and 9 during winter season were used for thorough histological study. In order to record histomorphometric Fig. 1. Paired testes weight in relation to natural photoperiod in data 10 photomicrographs were taken randomly from the different seasons. central zone of cross-sections of each testis using a Photomicroscope (AmScope Trinocular compound Morphometry of seminiferous tubule by season and day microscope +1.3 MP camera, Model T490 B-MT) and length. Morphometric evaluation of seminiferous tubules Toup View 3.0 image processing software. Using ImageJ revealed that in summer and rainy season they had their higher 1.45 image measurement software, diameters were diameter, circumference and germinal epithelium height than calculated from 20 randomly selected cross-sections of in autumn and winter (Figs. 2, 3, 4, 5, 6). Thus seminiferous the seminiferous tubules in each testis and recorded their tubules revealed a seasonal variation with two phases: (1) average values. Temperature and day length were recorded reproductive active phase, (2) regressive phase. Elevated from the official website (www.bmd.gov.bd) of values were observed in long day period (May - July) and Bangladesh Meteorological Department of Government decreased progressively until December. A new increase of the People's Republic of Bangladesh. occurred in February, March, followed by an increase in April, May, June and in July it reaches its peak and a progressive Data analysis. Student paired t-tests were used to com- decrease until December. The variation of the height of the pare between paired groups, whereas the one way analysis seminiferous epithelium in general accompanied the annual of variance (ANOVA) was used to compare the differences variation of the tubular diameter. Moderate peaks were in weight, volume, circumference and length of testes, observed in autumn (October, November) and maximum seminiferous tubule diameter, height of germinal values in summer and rainy season i.e. from March to epithelium across the groups. SPSS statistical software September (a marked peak). The lowest values were detected (Ver.16.0 for windows, SPSS) was used for analysis of in winter season (November, December and January). There the data. Results were expressed as arithmetic Mean ± Stan- was strong positive correlation (r=0.84) between dard Deviation (Mean±SD). The differences were seminiferous tubule diameter and paired testes weight considered to be significant if P was ≤0.05. throughout the year. Increase in testes weight also correlated 101 SHIL, S. K.; QUASEM, A. & RAHMAN, M. L. Histological and morphometric analyses of testes of adult quail (Coturnix coturnix japonica) of Bangladesh. Int. J. Morphol., 33(1):100-104, 2015.

well with the increase of height (r=0.88) of the germinal epithelium of the seminiferous tubule.

Fig. 2. Seasonal variation in histomorphometry of seminiferous tubule.

Fig. 3. Histological characters of seminiferous tubule in Summer Fig. 4. Histological characters of seminiferous tubule in Rainy (June) (scale bar 200 µm). season (August) (scale bar 200 µm).

Fig. 5. Histological characters of seminiferous tubule in Autumn Fig. 6. Histological characters of seminiferous tubule in Winter (October) (scale bar 200 mm) (January) (scale bar 200 mm). 102 SHIL, S. K.; QUASEM, A. & RAHMAN, M. L. Histological and morphometric analyses of testes of adult quail (Coturnix coturnix japonica) of Bangladesh. Int. J. Morphol., 33(1):100-104, 2015.

DISCUSSION

Gross Morphometry. Our study reveals that testes weight active phase (June - July), regressive phase (September - of quail varied significantly with the season. These results October) and non-active phase (January - February). Baraldi- partially agree with some previous findings (Haldar & Rai, Artoni et al. have found four distinct phases in annual cycle 1997; Haldar & Singh, 2001; Akbar et al., 2012) but the of Japanese quail in Brazil: a resting phase (at the end of exception is the inactive phase in winter. This observation summer), a recrudescence phase (in the fall), a proliferative confirmed the results of Akbar et al. in Japanese quails of phase (at the end of winter and beginning of spring) and a who reported the higher paired testicular weight regression phase (spring and summer). (4.53±0.370 g) in summer (sexually active phase) and lowest (0.78±0.048 g) in winter (inactive phase). In this study paired Effect of day length. In summer (March - June) and rainy testes weight of quail was found more than 1.0 g. season (July - September) day length ranges from 12 hours to Spermatogenic activity is closely related to testes weight 13 and half hours. But in autumn (October - November) and and in young quail it had to exceed 1.0 g for full winter (December - February) it ranges from 10 and half to 12 spermatogenic activity (Wilson, 1972). Higher relative paired hours in Bangladesh. Baraldi-Artoni et al. also found elevated testes weigh in summer season indicates their reproductive and maximum spermatogenic activity in fall-winter (short- active phase and lower in autumn and winter indicates their day period) and at the beginning of spring, respectively and regressive phase. lower activity during spring and summer (long-day periods) with minimum values at the end of summer. Boon et al. (2000) Histomorphometry. In the present study, the spermatogenic and Mills et al. (1997) found that in Japanese quail sexual activity of the domestic quail was evaluated on the basis of development is depend on the length of the daily light period: the diameter of seminiferous tubules and germinal epithelium long photoperiods stimulate sexual maturation, while short height. We found that seminiferous tubule diameter, height of photoperiods inhibit or delay sexual maturation. They found the germinal epithelium and circumference of the cross section increased testicular weight and volume in long day period but of the seminiferous tubule were significantly (P<0.05) higher in contrast, growth under or transfer of sexually developed in summer and rainy season (March to September) as compared birds to short days was found regression of gonadal with autumn and winter (October to February). Though the development and function. Light shift experiments have also height of the germinal epithelium were lower in autumn and demonstrated that transfer of such sexually regressed males winter (57.5±2.5 µm and 61.0±8.54 µm respectively), mature back to long day results in re growth and restored function of spermatozoa were found in the lumen of seminiferous tubule the testes (Domjan, 1987; Chaturvedi et al., 1992). Oishi & throughout the year. Study on quail in Brazil of southern Konishi (1983), Delville et al. (1984) and Domjan they have hemisphere of the world, Baraldi-Artoni et al. (2007) observed also found the fully developed gonad of quail in long day the moderate peaks values of seminiferous tubule diameter in period. January, April and May and maximum values in September (a marked peak). The lowest values were detected in March, August and December. Highest and lowest values were 155 CONCLUSION µm and 75 µm respectively which differ with findings of present study. In our findings, mature spermatozoa in the lumen indicate that their reproductive efficiency throughout the year It is suggested that the degree of gonadal growth in but have two phases in its annual reproductive cycle: sexually quail depends on the availability of daily photoperiod. Arti- active phase stays throughout the period of summer and rainy ficial illumination in short day period (autumn and winter) season and regressed phase in autumn and winter. These results may keep quail sexually active throughout the year. agreed with previous findings (Haldar & Rai; Haldar & Singh) but differ in some points. They have found in this tropical environment () that the Indian jungle bush quail ACKNOWLEDGEMENTS ( asiatica) had three distinct phases reproductive ac- tive phase in summer, regressive phase in autumn and non- active phase in winter. In Bangladesh, winter does not show Authors are grateful to Danish International an extreme weather in terms of temperature and day length, Developing Agency (DANIDA) funded Poultry Research so this may be the cause for not finding the sexually inactive and Training Center (PRTC) for financial support. Authors phase of testis in winter (December - February). Akbar et al. also express their highest gratitude to Prof. Dr. Md. Ahasanul found that Japanese quails in Pakistan showed reproductive Haque for his valuable suggestions. 103 SHIL, S. K.; QUASEM, A. & RAHMAN, M. L. Histological and morphometric analyses of testes of adult quail (Coturnix coturnix japonica) of Bangladesh. Int. J. Morphol., 33(1):100-104, 2015.

SHIL, S. K.; QUASEM, A. & RAHMAN, M. L. Análisis histológico y morfométrico de testiculos de codorniz adulta (Coturnix coturnix japonica) de Bangladesh. Int. J. Morphol., 33(1):100-104, 2015.

RESUMEN: Se realizó un estudio histológico y morfométrico para evaluar las variaciones testiculares estacionales de la codorniz japo- nesa adulta (Coturnix coturnix japonica) en Bangladesh. Se midieron el peso de las aves y sus testículos; el volumen, longitud, circunferencia y diámetro de los túbulos seminíferos, y la circunferencia y altura del epitelio germinal testicular. Secciones de testículos obtenidos de las aves estudiadas fueron teñidas con H&E y las imágenes fueron analizadas con el programa ImageJ. Se registró un aumento significativo en peso y volumen de los testículos de codornices (P<0,05) en el periodo día de verano y en la temporada de lluvia (marzo-septiembre) al período del día corto de otoño e invierno (octubre-febrero). El período estacionario no tuvo efecto significativo sobre el aumento de peso vivo de las codornices. El peso y el diámetro de los testículos, y la altura del epitelio germinal del túbulo seminífero se correlacionaron positivamente (r=0,84; r=0,88). La actividad espermatogénica fue más pronunciada durante el verano y durante la temporada de lluvias que en otoño e invierno (p<0,05).

PALABRAS CLAVE: Codorniz japonesa; Morfometría; Testículo; Estación; Duración del día; Temperatura.

REFERENCES Haldar, C. & Singh, S. S. Melatonin and immunological functions/expression by the bursa of Fabricius in Indian jungle bush quail (Perdicula asiatica). Avian Endocrinol., 1:427-35, 2001. Akbar, Z.; Qureshi, A. S. & Rahman. S. U. Effects of seasonal variation in different reproductive phases on the cellular response of bursa and tes- Lake, P. E. Male genital organs. In: Kim, A. S. & McLelland, J. (Eds.). Form tes in Japanese quails (Coturnix japonica). Pak. Vet. J., 32(4):525-9, and function in birds. London, Academic Press, 1981. pp. 1-61. 2012. Lofts, B. & Murton, R. K. Photoperiodic and physiological adaptations Ball, G. F. The neural integration of environmental information by seasonally regulating avian breeding cycles and their ecological significance. J. breeding birds. Am. Zool., 33:185-99, 1993. Zool., Lond., 155(3):327-94, 1968.

Baraldi-Artoni, S. M.; Bottino, F.; Oliveira, D.; Sobue Franzo, V.; Amoroso, Mills, A. D.; Crawford, L. L.; Domjan, M. & Faure, J. M. The behavior of the L.; Orsi, A. M. & Cruz, C. Morphometric study of Rynchotus rufescens Japanese or domestic quail Coturnix japonica. Neurosci. Biobehav. Rev., testis throughout the year. Braz. J. Biol., 67(2):363-7, 2007. 21(3):261-81, 1997.

Bath, G. S. & Chaudhari, S. U. R. Sperm reserves and its relationship to Oishi, T. & Konishi, T. Variations in the photoperiodic cloacal response of parameters of the testis, epididymis and vas deferens of local cocks in Japanese quail: association with testes weight and feather color. Gen. the Sahel Region of Nigeria. Int. J. Agric. Biol., 4(4):561-4, 2002. Comp. Endocrinol., 50(1):1-10, 1983.

Boon, P.; Visser, G. H. & Daan, S. Effect of photoperiod on body weight Orlu, E. E. & Egbunike, G. N. Breed and seasonal variations in the testicular gain, and daily energy intake and energy expenditure in Japanese quail morphometry, gonadal and extragonadal sperm reserves of the barred (Coturnix c. japonica). Physiol. Behav., 70(3-4):249-60, 2000. plymouth rock and Nigerian indigenous breeds of the domestic fowl. Pak. J. Biol. Sci., 13(3):120-5, 2010. Chaturvedi, C. M.; Dubey, L. & Phillips, D. Influence of different photoperiods on development of gonad, cloacal gland and circulating thyroid hormones Wilson, W. O. A review of the physiology of coturnix (Japanese quail). World in male Japanese quail Coturnix coturnix japonica. Indian J. Exp. Biol., Poult. Sci. J., 28(4):413-429, 1972. 30(8):680-4, 1992. Wilson, W. O.; Cain, J. R. & Siopes, T. D. The interaction of light and Cockrem, J. F. Timing of seasonal breeding in birds, with particular reference temperature in Coturnix. Poult. Sci., 50(1):204-7, 1971. to New Zealand birds. Reprod. Fert. Dev., 7(1):1-19, 1995. Wingfield, J. C.; Hahn, T. P.; Levin, R. & Honey, P. Environmental Delville, Y.; Hendrick, J. C.; Sulon, J. & Balthazart, J. Testosterone metabolism predictability and control of gonadal cycles in birds. J. Exp. Zool., and testosterone-dependent characteristics in Japanese quail. Physiol. 261:214-31, 1992. Behav., 33(5):817-23, 1984. Correspondence to: Domjan, M. Photoperiodic and endocrine control of social proximity behavior Subrata Kumar Shil, DVM, MS in male Japanese quail (Coturnix coturnix japonica). Behav. Neurosci., Assistant Professor 101(3):385-92, 1987. Department of Anatomy and Histology Follett, B. K. & Farner, D. S. Pituitary gonadotropins in the Japanese quail Chittagong Veterinary and Animal Sciences University (Coturnix coturnix japonica) during photoperiodically induced gonadal Khulshi-4202 growth. Gen. Comp. Endocrinol., 7:125-31, 1966. Chittagong - BANGLADESH Hahn, T. P.; Boswell, T.; Wingfield, J. C. & Ball, G. F. Temporal flexibility in avian reproduction. In: Nolan, V. Jr.; Ketterson, E. D. & Thompson, C. Email: [email protected] F. (Eds.). Current Ornithology. New York, Plenum, 1997. pp.9-80. Haldar, C. & Rai, V. Photoperiod, indoleamines, and ovarian responses in the Indian tropical jungle bush quail Perdicula asiatica. J. Exp. Zool., Received: 19-05-2014 277(6):442-9, 1997. Accepted: 05-11-2014 104